Mitochondrial 12S rRNA A827G mutation is involved in the genetic susceptibility to aminoglycoside ototoxicity

We have analyzed the clinical and molecular characterization of a Chinese family with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluations revealed that only those family members who had a history of exposure to aminoglycoside antibiotics subsequently developed hearing loss, suggesting mitochondrial genome involvement. Sequence analysis of the mitochondrial 12S rRNA and tRNA(Ser(UCN)) genes led to the identification of a homoplasmic A827G mutation in all maternal relatives, a mutation that was identified previously in a few sporadic patients and in another Chinese family with non-syndromic deafness. The pathogenicity of the A827G mutation is strongly supported by the occurrence of the same mutation in two independent families and several genetically unrelated subjects. The A827G mutation is located at the A-site of the mitochondrial 12S rRNA gene which is highly conserved in mammals. It is possible that the alteration of the tertiary or quaternary structure of this rRNA by the A827G mutation may lead to mitochondrial dysfunction, thereby playing a role in the pathogenesis of hearing loss and aminoglycoside hypersensitivity. However, incomplete penetrance of hearing impairment indicates that the A827G mutation itself is not sufficient to produce clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Indeed, aminoglycosides may contribute to the phenotypic manifestation of the A827G mutation in this family. In contrast with the congenital or early-onset hearing impairment in another Chinese family carrying the A827G mutation, three patients in this pedigree developed hearing loss only after use of aminoglycosides. This discrepancy likely reflects the difference of genetic backgrounds, either mitochondrial haplotypes or nuclear modifier genes, between two families.     

An integrated morphological and molecular approach to a new species description in the Trypanosomatidae: the case of Leptomonas podlipaevi n. sp., a parasite of Boisea rubrolineata (Hemiptera: Rhopalidae)

Leptomonas podlipaevi n. sp., a new trypanosomatid species, is described herein based on light microscopic, ultrastructural, and molecular phylogenetic data. The organism is pleomorphic both in host and culture, with two predominant forms-a typical promastigote with a long flagellum and a shorter promastigote with a small or barely extending flagellum. Several spliced leader RNA repeat sequences obtained from the original cultures and the clonal lines representing two types of cells were all nearly identical. These sequences formed a tight cluster in the neighbor-joining tree well separated from other trypanosomatid species. Glyceraldehyde phosphate dehydrogenase gene sequences were determined for L. podlipaevi and 10 previously described trypanosomatid species. Molecular phylogenetic analysis has demonstrated that the new species is most closely related to Leptomonas seymouri and Leptomonas pyrrhocoris. The analysis has also highlighted the polyphyly of the genus Leptomonas.     

A platform of high-efficiency nonviral gene transfer in mouse osteoblast cells in vitro

We have previously established mouse genetic models and identified the genetic components of quantitative trait loci (QTL) on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and bone's anabolic response to mechanical loading. However, these regions contain dozens of unknown genes that are needed for functional testing. In this study, we provided a protocol of nucleoporation with high efficiency by using a commercial nucleofection buffer and Gene Pulser to deliver a test gene into bone cells for functional studies. We cloned an osteoblast differentiation-specific geneosterix (Osx) from a mouse bone cDNA library into a pHGCX expression vector and used nucleoporation to deliver pHGCX/Flag-Osx into the nuclei of MC3T3-E1 cells. We then examined the transfection efficiency transgene expression, and function. Our results have demonstrated that nucleoporation can deliver a transgene into MC3T3-E1 osteoblast cells with approx 94% transfection efficiency, and express a functional Flag-Osx fusion protein capable of inducing cell differentiation as measured by an incease in alkaline phosphatase (ALP) activity. Therefore, this experimental system provides a rapid, safe, and efficient cell-based model of high-throughput phenotypic screening to identify candidate genes from physically mapped regions that are important for osteoblast differentiation.     

Ex vivo expanded umbilical cord blood T cells maintain naive phenotype and TCR diversity

BACKGROUND: Umbilical cord blood (CB) is a promising source of hematopoietic stem cells for allogeneic transplantation. However, delayed engraftment and impaired immune reconstitution remain major limitations. Enrichment of donor grafts with CB T cells expanded ex vivo might facilitate improved T-cell immune reconstitution post-transplant. We hypothesized that CB T cells could be expanded using paramagnetic microbeads covalently linked to anti-CD3 and anti-CD28 Ab. METHODS: CB units were divided into three fractions: (1) cells cultured without beads, (2) cells cultured with beads and (3) cells cultured with beads following CD3+ magnetic enrichment. All fractions were cultured for 14 days in the presence of IL-2 (200 IU/mL). RESULTS: A mean 100-fold expansion (range 49-154) of total nucleated cells was observed in the CD3+ magnetically enriched fraction. Following expansion, CB T cells retained a naive and/or central memory phenotype and contained a polyclonal TCR diversity demonstrated by spectratyping. DISCUSSION: Our data provide evidence that naive and diverse CB T cells may be expanded ex vivo and warrant additional studies in the setting of human CB transplantation.     

Application of cell engineering technology to the tumour immunotherapeutic drug: a review

The concept of cell engineering is frequently mentioned in recent years. It's a comprehensive technology which is very powerful and useful. By using it, scientists can produce many kinds of proteins and other biological moleculars in large scale that are in great demand for the human being. Among these products, most can be used for tumor immunotherapy and have been confirmed to be very effective in tumour control or subsidiary treatment. Of them, most common and useful drugs include three sorts; tumour vaccines, cell factors and monoclonal antibodies. The paper will discuss in detail related drugs application advances in tumour immunotherapy.     

Characterization of nuclear import and export signals determining the subcellular localization of WD repeat-containing protein 42A (WDR42A)

WD repeat-containing protein 42A (WDR42A) is a member of the WD40-repeat proteins. Here, we investigated the localization pattern of WDR42A in living cells. By mutational analysis, a nuclear localization signal, 114PRRRVQRKR122, was for the first time determined. The dominant negative, co-immunoprecipitation and GST pull-down results further demonstrated that the nuclear import of WDR42A was mediated by karyopherin-α1/β1 in conjunction with the GTPase Ran. Additionally, a nuclear export signal, 39IEVEASDLSLSL50, was verified to be a functional NES, which mediated the nuclear export through Chromosome Region Maintenance 1 dependent pathway. All these data suggest WDR42A is a nucleocytoplasmic shuttling protein.     

Downregulation of galectin-3 by EGF mediates the apoptosis of HepG2 cells

Epidermal growth factor (EGF) in high concentrations induces apoptosis of the tumor cells which express high levels of epidermal growth factor receptor. However, the precise mechanism for this induction is not clear. Galectin-3 is the most probable candidate for mediating this effect, as it is known to induce anti-apoptotic activity in a variety of tumor cells exposed to diverse apoptotic stimuli. In this study, we determined whether galectin-3 plays a role in high concentrations of EGF-induced apoptosis of HepG2 cells. We found that EGF in high concentrations led to the growth inhibition of HepG2 cells, which were associated with promotion of cell death. High concentrations of EGF suppressed cytoplasmic expression of galectin-3. Moreover, we demonstrated overexpression of galectin-3 could reduce EGF-induced apoptosis in HepG2 cells. Our study demonstrated for the first time that downregulation of cytoplasmic galectin-3 was essential for high concentrations of EGF-induced apoptosis in HepG2 cells.     

A novel single nucleotide polymorphism in exon 7 of LPL gene and its association with carcass traits and visceral fat deposition in yak (Bos grunniens) steers

Lipoprotein lipase (LPL) is considered as a key enzyme in the lipid deposition and metabolism in tissues. It is assumed to be a major candidate gene for genetic markers in lipid deposition. Therefore, the polymorphisms of the LPL gene and associations with carcass traits and viscera fat content were examined in 398 individuals from five yak (Bos grunniens) breeds using PCR–SSCP analysis and DNA sequencing. A novel nucleotide polymorphism (SNP)-C→T (nt19913) was identified located in exon 7 in the coding region of the LPL gene, which replacement was responsible for a Phe-to-Ser substitution at amino acid. Two alleles (A and B) and three genotypes designed as AA, AB and BB were detected in the PCR products. The frequencies of allele A were 0.7928, 0.7421, 0.7357, 0.6900 and 0.7083 for Tianzhu white yak (WY), Gannan yak (GY), Qinghai-Plateau yak (PY), Xinjiang yak (XY) and Datong yak (DY), respectively. The SNP loci was in Hardy–Weinberg equilibrium in five yak populations (P > 0.05). Polymorphism of LPL gene was shown to be associated with carcass traits and lipid deposition. Least squares analysis revealed that there was a significant effect on live-weight (LW) (P < 0.01), average daily weight gain (ADG) and carcass weight (P < 0.05). Individuals with genotype BB had lower mean values than those with genotype AA and AB for loin eye area and viscera fat weight (% of LW) in 25–36 months (P < 0.05). The results indicated that LPL gene is a strong candidate gene that affects carcass traits and fat deposition in yak.